The material producing method relating to the culture of microorganisms or culture cells is mainly classified into (1) Batch culture method and Fed-Batch culture method, as well as (2) continuous fermentation method.
In the above-mentioned Batch culture method and Fed-Batch culture method of (1), there are advantages in which culture can be completed using only a simple facility in a short time, and little damage is caused by bacterial contamination. For this reason, these methods have been conventionally used as a substance producing method utilizing microorganisms or culture cells. However, in those methods, since the concentration of fermentation product in a culture liquid becomes higher with an elapse of the time, the productivity and yield are lowered, for example, by an increase of the osmotic pressure or inhibition of the fermentation due to the product itself. For those reasons, these culture methods make it difficult to maintain the productivity and yield of a fermentation product in a high level stably for a long time.
On the other hand, the continuous fermentation method of the above-mentioned (2) is characterized in that, by avoiding the fermentation product in a fermentation tank from accumulating with a high concentration, the productivity and yield can be maintained in a high level for a long time.
For example, a continuous fermentation method has been disclosed with respect to the fermentation of L-glutamic acid (see JP-A No. 10-150996) and L-lysine (see Toshihiko Hirao et al., Appl. Microbiol. Biotechnol. 32, 269 273 (1989)). However, in these examples, although materials such as nutrients are continuously supplied to a culture liquid, the culture liquid containing microorganisms or culture cells is also drawn, with the result that the microorganisms or culture cells in the culture liquid are diluted; therefore, the improvement of its production efficiency is limited.
For this reason, as the continuous fermentation method, a method has been proposed in which microorganisms or culture cells are filtered through a separation membrane, and while the fermentation product is collected from a filtration liquid, the filtered microorganisms or culture cells are held in the fermentation tank or refluxed thereto to maintain the concentration of the microorganisms or cells in the culture liquid in a high level.
For example, a technique has been proposed in which continuous fermentation is carried out by using a continuous fermentation apparatus with a separation membrane (see International Publication No. 07/097260 Pamphlet). In this proposal, a continuous fermentation apparatus provided with a tank used for cultivating microorganisms or culture cells and a tank used for membrane separation on a target fermentation product from the microorganisms and culture cells in the culture liquid, is used so that various chemical products can be produced at a higher production speed in comparison with the batch culture method and with the fed-batch culture method.
In the continuous fermentation apparatus utilizing a separation membrane, it is thought that improving the flow velocity of culture liquid inside a membrane separation tank leads to make the membrane less fouling; as a result, the production speed can be improved due to increase in the quantity of filtration liquid through the separation membrane.
In WO '260, however, since the liquid transfer quantity from the fermentation tank and the flowing quantity into the membrane separation tank cannot be controlled separately, the flowing quantity of the culture liquid to be supplied to the membrane separation tank depends on the flowing quantity of the culture liquid transferred from the fermentation tank. Therefore, in an attempt to change the flow velocity of the culture liquid inside the membrane separation tank, the liquid transfer quantity from the fermentation tank needs to be changed, with the result that a liquid mixing state inside the fermentation tank is changed to cause serious changes of culture conditions. Moreover, when a pressure inside the membrane separation tank was increased due to fouling of the membrane or an increase in the concentration of the microorganisms or culture cells with an elapse of the time, and the like, it is preferable to reduce the flowing quantity of the culture liquid to be supplied to the membrane separation tank to optimize the membrane separation itself. However, when the flowing quantity of the culture liquid to be supplied to the membrane separation tank is changed, the culture conditions in the fermentation tank are changed greatly. For this reason, the flowing quantity of the culture liquid to be supplied to the membrane separation tank cannot be changed easily. In addition, when the quantity of culture liquid to be transferred from the fermentation tank is reduced to optimally control the pressure inside the membrane separation tank, the flow velocity of the culture liquid inside a liquid transfer line is decreased and the microorganisms or culture cells are precipitated inside the liquid transfer line, and a problem of decreasing of the production efficiency occurs. In contrast, when the pressure inside the membrane separation tank is too high, the microorganisms in the culture liquid transferred outside from the membrane separation tank might be damaged due to pressure fluctuation.
It could therefore be helpful to provide a method of producing a chemical product that can control flow velocity of a culture liquid inside a membrane separation tank without giving influences to culture conditions in the fermentation tank, and also suppress precipitation of microorganisms or culture cells so that the production efficiency of the chemical product can be improved, as well as a fermentation apparatus to which such a method can be desirably applied.
We found that by using any of the following structures (1) to (14), it is possible to properly maintain culture conditions (retention time of the culture liquid and so on), while controlling the flow velocity of culture liquid inside a membrane separation tank, and consequently to efficiently produce a chemical product, and complete this disclosure.                (1) A method of producing a chemical product including the steps of: cultivating microorganisms or culture cells in a fermentation tank; transferring a culture liquid from the fermentation tank to a membrane separation tank to filter the culture liquid through a separation membrane; and collecting a fermentation product from a filtration liquid as the chemical product while refluxing an unfiltered culture liquid that has not been filtered to be joined to the culture liquid on an upstream side of the membrane separation tank, wherein one portion of the culture liquid to be transferred from the fermentation tank is allowed to bypass the membrane separation tank depending on a pressure at the culture liquid flow-in side of the membrane separation tank.        (2) The method of producing a chemical product according to the above-mentioned (1), in which a flowing quantity of the culture liquid to be allowed to bypass the membrane separation tank is controlled so that a gauge pressure at the culture liquid flow-in side of the membrane separation tank is 1 MPa or less.        (3) The method of producing a chemical product according to the above-mentioned (1) or (2), in which one portion of the unfiltered culture liquid is refluxed to be joined to the culture liquid in the fermentation tank, while the rest of the portion of the unfiltered culture liquid is refluxed to be joined to a culture liquid located between the fermentation tank and the membrane separation tank.        (4) The method of producing a chemical product according to the above-mentioned (3), in which a flowing quantity of the unfiltered culture liquid to be refluxed to be joined to the culture liquid located between the fermentation tank and the membrane separation tank and a flowing quantity of the unfiltered culture liquid to be refluxed to be joined to the culture liquid in the fermentation tank are each independently controlled.        (5) The method of producing a chemical product according to the above-mentioned (3) or (4), in which a ratio of a flowing quantity of the unfiltered culture liquid to be refluxed to be joined to the culture liquid in the fermentation tank to a flowing quantity of the unfiltered culture liquid to be refluxed to be joined to the culture liquid located between the fermentation tank and the membrane separation tank is 1 or less.        (6) The method of producing a chemical product according to any one of the above-mentioned (1) to (5), in which each of the linear speed of the culture liquid to be transported from the fermentation tank to the membrane separation tank, the linear speed of the unfiltered culture liquid that is refluxed from the membrane separation tank to be joined to the culture liquid on the upstream side of the membrane separation tank and the linear speed of the culture liquid that is allowed to bypass the membrane separation tank is 2.5 cm/sec or more.        (7) The method of producing a chemical product according to any one of the above-mentioned (1) to (6), in which a quantity of culture liquid to flow into the membrane separation tank and/or a quantity of filtration liquid from the separation membrane are adjusted so that the recovery percentage of the quantity of filtration liquid from the separation membrane to the quantity of culture liquid to flow into the membrane separation tank is 10.0% or less.        (8) The method of producing a chemical product according to any one of the above-mentioned (1) to (7), in which a ratio of the culture liquid volume in the fermentation tank to a culture liquid volume in the membrane separation tank is 4 or more to 100 or less.        (9) A continuous fermentation apparatus including: a fermentation tank for cultivating microorganisms or culture cells; a membrane separation tank having a separation membrane used for collecting a fermentation product produced in a culture liquid from the fermentation tank; a circulation line that connects the fermentation tank with the membrane separation tank to transfer the culture liquid to the membrane separation tank, and refluxes an unfiltered culture liquid that has not been filtered through the separation membrane to be joined to the culture liquid on the upstream side of the membrane separation tank; and a culture liquid transfer means installed in the circulation line, in which this structure further includes a bypass line for the membrane separation tank; a detection means for a pressure at the culture liquid flow-in side of the membrane separation tank; and a flowing quantity control means installed in the bypass line.        (10) The continuous fermentation apparatus according to the above-mentioned (9), in which the flowing quantity control means is operated in response to the detection result of the detection means.        (11) The continuous fermentation apparatus according to the above-mentioned (9) or (10), further including a linear speed detection means for the circulation line so that the flowing quantity control means and/or the culture liquid transfer means are operated in response to the detection result of the linear speed detection means.        (12) The continuous fermentation apparatus according to any one of the above-mentioned (9) to (11), in which the membrane separation tank is set up in a circulation circuit having a liquid transfer means different from the culture liquid transfer means, which is independent from the fermentation tank.        (13) The continuous fermentation apparatus according to any one of the above-mentioned (9) to (12), in which the circulation line has an opening at a position that is immersed with the culture liquid to be stored in the fermentation tank.        (14) The continuous fermentation apparatus according to any one of the above-mentioned (9) to (13), in which the ratio of the fermentation tank volume to the membrane separation tank volume is from 4 or more to 100 or less.        
One portion of the culture liquid to be transferred from the fermentation tank is allowed to bypass the membrane separation tank depending on a pressure at the culture liquid flow-in side of the membrane separation tank, that is, the flowing quantity of the culture liquid to be supplied to the membrane separation tank and the flowing quantity of the culture liquid to be transferred from the fermentation tank can be controlled independently. As a result, it is possible to make fouling of the membrane hardly occur, by appropriately changing the flow velocity of the culture liquid inside the membrane separation tank without changing the culture conditions, and consequently to increase the quantity of filtration liquid and improve the producing speed. Even if fouling of the membrane occurs with an elapse of the time or the concentration of the microorganisms or culture cells increases to cause a pressure rise inside the membrane separation tank, it is possible to transfer the culture liquid to the membrane separation tank, without causing virtually any change of culture conditions in the fermentation tank, and also to control the flowing quantity of the culture liquid to be supplied to the membrane separation tank and the pressure exerted in the membrane separation tank, while maintaining a flow velocity that hardly causes the microorganisms or culture cells to precipitate in the circulation line used for refluxing the unfiltered culture liquid that has not been filtered by the separation membrane, and as a result, it becomes possible to prevent damages to the membrane separation tank and also to prevent destruction of the microorganisms and culture cells in the culture liquid due to pressure fluctuations. Moreover, even upon occurrence of a failure inside the membrane separation tank, it is possible to completely stop the supply of the culture liquid into the membrane separation tank and correct the failure inside the membrane separation tank, or to exchange or switch membrane separation tanks while the fermentation is being continuously carried out.
Moreover, by controlling the recovery percentage of the filtration liquid in the membrane separation tank to 10% or less, with one portion of the culture liquid to be transferred from the fermentation tank being allowed to bypass the membrane separation tank depending on the pressure at the culture liquid flow-in side of the membrane separation tank, it becomes possible to further prevent fouling of the membrane and to prolong a continuous fermentation time.
As described above, production efficiency and sugar-related yield of a fermentation product obtained by continuous fermentation (that is, a desired product) can be simultaneously improved, and by further controlling the recovery percentage in the membrane separation tank to 10% or less, the continuous fermentation time can be also prolonged.